Enhanced Immunogenicity For GPI-anchored Antigens

ABSTRACT

Compositions and methods are presented that allow for an enhanced immune response against a GPI-anchored tumor associated antigen by modification of the protein portion of the TAA to include a transmembrane domain and a trafficking signal that directs the modified protein to the endosomal or lysosomal compartment. Most preferably, the modified protein will no longer have a GPI anchor or GPI attachment sequence.

This application claims priority to our copending U.S. provisionalapplication with the Ser. No. 62/618,087, which was filed Jan. 17, 2018.

FIELD OF THE INVENTION

The field of the invention is compositions and methods of enhancedimmunogenicity for antigens, especially as it relates to variouscompositions and methods that enhance the immunogenicity of GPI-anchoredantigens in cancer therapy.

BACKGROUND OF THE INVENTION

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.

Significant improvements have been made in cancer immunotherapiestargeting patient- and tumor specific antigens (neoepitopes). Whilepromising, identification of suitable antigens and subsequent creationof tailored therapeutic compositions is time consuming and expensive.Alternatively, immune therapy can also target antigens that are commonto specific tumors (i.e., tumor associated antigens (TAA)). However, notall TAAs are equally effective across a diverse group of patients, andseveral TAAs tend to be entirely ineffective as an immunogenic entitydue to glycophosphatidylinositol (GPI) anchoring to the cell membrane,and FIG. 1 schematically illustrates the structure of a GPI anchor.

Unfortunately, various TAAs are GPI-anchored proteins and especiallyinclude CEA (carcinoembryonic antigen, often associated with epithelialcancers), PSCA (prostate stem cell antigen), mesothelin (oftenassociated with mesothelioma, ovarian and pancreatic adenocarcinoma),and urokinase plasminogen activator receptor (often associated withaggressive cancer growth and metastasis in many cancers, for example,gastric cancer) and as such will typically not be effective targets forcancer immune therapy.

While membrane anchors could conceptually be removed from a protein,thusly modified proteins will in most circumstances not be translocatedto the cell surface and so fail to exert proper function. On the otherhand, certain phospholipid anchors of a membrane anchored glycoproteincan be replaced with a transmembrane domain as was shown for slime moldDictyostelium discoideum. However, such replacement substantiallyreduced residence time on the cell surface (Cell Biol., Volume 124,Numbers 1 & 2, January 1994 205-215), and so modified proteins aretherefore less likely to be bound by an antibody.

Thus, even though various TAAs may conceptually serve as therapeutictargets common to many cancers and as such may eliminate the need forpersonalized therapy, not all of the TAA are sufficiently immunogenic.Consequently, there is a need to provide improved compositions andmethods that enhance immunogenicity of TAA, and especially GPI-anchoredTAA.

SUMMARY OF THE INVENTION

The inventive subject matter is directed to various immune therapeuticcompositions and methods in which GPI-anchored antigens are modifiedsuch that the modified antigen will no longer be coupled to a GPIportion but instead include one or more transmembrane domains and acytoplasmic tail sequence derived or adapted from a protein thattraffics the antigen to the endosomal or lysosomal system (e.g. CD1a,CD1c, Lamp1 portion). Such modified antigens resulted in increased CD4+stimulation (relative to unmodified GPI-anchored antigen) and alsoresulted in increased stimulation of (polyfunctional) CD8+ T cells.

In one aspect of the inventive subject matter, the inventor contemplatesa recombinant hybrid protein that comprises an antigen portion that iscoupled to at least one transmembrane domain and a trafficking element.In preferred aspects, the antigen portion is at least a portion of aGPI-anchored protein, and the trafficking element directs therecombinant hybrid protein to a sub-cellular location (typicallyrecycling endosome, sorting endosome, or lysosome).

For example, suitable GPI-anchored proteins are TAAs and especiallyinclude CEA, PSCA, mesothelin, and the urokinase plasminogen activatorreceptor, as well as non-cancer disease associated protein (e.g.,variant surface protein of Trypanosoma brucei or a prion protein). Inother examples, the antigen portion lacks a functional GPI anchoringsignal, and/or the transmembrane domain comprises at least a portion ofa transmembrane domain of an alpha, beta, or zeta chain of a T-cellreceptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137,CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB(CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160,CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4,CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a,LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1,ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CEACAM1, CRTAM,Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108),SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, orPAG/Cbp.

While not limiting to the inventive subject matter, the transmembranedomain is preferably bound in-frame to the C-terminus of the antigenportion (with or without a peptide linker), and most preferably includesan endosomal trafficking element of CD1a, CD1c, or Lamp 1. Furthermore,it is generally preferred that the trafficking element is bound in-frameto the C-terminus of the transmembrane domain.

In a further contemplated aspect of the inventive subject matter, theinventor also contemplates a recombinant nucleic acid that comprises asequence segment encoding the hybrid protein as contemplated herein,operably linked to a promoter to drive expression of the hybrid protein.Most typically, the recombinant nucleic acid is a viral expressionvector (e.g., an adenoviral expression vector, preferably having adeleted E1 and E2b gene), and the promoter is a constitutive promoter oran inducible promoter (e.g., inducible by hypoxia, IFN-gamma, or IL-8).In addition, contemplated recombinant nucleic acids may further comprisea sequence that encodes at least one of a co-stimulatory molecule, animmune stimulatory cytokine, a protein that interferes with ordown-regulates checkpoint inhibition, and an adjuvant polypeptide.

Suitable co-stimulatory molecules include OX4OL, 4-1BBL, CD80, CD86,CD30, CD40, CD3OL, CD4OL, ICOS-L, B7-H3, B7-H4, CD70, GITR-L, TIM-3,TIM-4, CD48, CD58, TL1A, ICAM-1, and LFA3, while contemplated immunestimulatory cytokines include IL-2, IL-12, IL-15, IL-15 super agonist(ALT803), IL-21, IPS1, and LMP1, and suitable proteins that interfereinclude an antibody or an antagonist of CTLA-4, PD-1, TIM1 receptor,2B4, or CD160. Contemplated adjuvant polypeptides include calreticulinor a portion with adjuvant activity thereof, or HMGB1 or a portion withadjuvant activity thereof.

Viewed from a different perspective, the inventor also contemplatesrecombinant virus comprising the recombinant nucleic acid presentedherein, and especially a replication deficient virus (e.g., adenovirushaving a deleted E1 and E2b gene). Likewise, recombinant antigenpresenting cell are also contemplated that comprise the recombinantnucleic acids presented herein.

In yet another aspect of the inventive subject matter, the inventor alsocontemplates a method of increasing antigenicity of a GPI-anchoredprotein that includes a step of modifying the protein portion of theGPI-anchored protein to include at least one transmembrane domain and atrafficking element, wherein the trafficking element directs themodified protein to a sub-cellular location (e.g., recycling endosome,sorting endosome, or lysosome). With respect to the GPI-anchored proteinit is preferred that the protein is a TAA (such as CEA, PSCA,mesothelin, or urokinase plasminogen activator receptor) or that theprotein is a non-cancer disease associated protein, optionally a variantsurface protein of Trypanosoma brucei or a prion protein.

Therefore, the inventor also contemplates a method of treating a tumorexpressing a GPI-anchored tumor associated antigen that includes a stepof administering a cell-based vaccine composition comprisingcontemplated recombinant hybrid proteins, or a step of administering aDNA or RNA-based vaccine composition that comprises contemplatedrecombinant nucleic acids.

For example, suitable cell-based vaccine compositions may comprise aplurality of recombinant autologous cells of the patient (preferablyantigen presenting cells), or may comprise recombinant yeast orbacterial cells. Similarly, the DNA or RNA-based vaccine composition maycomprise a recombinant adenovirus. Thus, contemplated pharmaceuticalcompositions may include a recombinant virus or a recombinant antigenpresenting cell as presented herein (typically formulated fortransfusion). Viewed from a different perspective, the inventor alsocontemplates the use of a recombinant virus as presented herein in thetreatment of cancer, and the use of a recombinant nucleic acid aspresented herein in the manufacture of a vaccine composition fortreatment of cancer.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic illustration of an exemplary GPI-anchored protein.

FIG. 2A is a schematic illustration of an exemplary immunizationschedule/schematic according to the inventive subject matter.

FIG. 2B is an exemplary schematic of recombinant constructs for modifiedCEA.

FIG. 3 shows exemplary results for CD4+ splenocytes following theimmunization schedule of FIG. 2A.

FIG. 4 shows exemplary results for CD8+ splenocytes following theimmunization schedule of FIG. 2A.

FIG. 5 shows exemplary ELISA results following the immunization scheduleof FIG. 2A.

DETAILED DESCRIPTION

The inventor has now discovered that GPI-anchored membrane proteins, andespecially disease associated GPI-anchored membrane proteins, can begenetically modified to so enhance their immunogenicity and render suchantigens suitable targets for immune therapy. Notably, by modificationof the proteins to drive them into the MHC II pathway, robust CD4+ cellactivation can be achieved, enabling cross-presentation, and support ofCD8+ cell growth via CD4 “helper” function. Such approach is especiallyadvantageous where the GPI-anchored membrane protein is a TAA such asCEA, which is heavily prevalent on epithelial cancers. Due to the GPIanchor, CEA is generally not a therapeutically effective antigen forstimulating CD8 T cells in its natural configuration. However,modification of GPI-anchored membrane proteins as presented hereinsubstantially increases immunogenicity and may thus render GPI-anchoredmembrane proteins therapeutic targets.

For example, and as discussed in more detail below, by replacing the GPIanchor on CEA with a transmembrane domain and cytoplasmic tail sequencesderived from proteins which enter the endosomal system (e.g., CD1a,CD1c, Lamp1), the inventor demonstrated that such modified proteinsachieve increased CD4 T cell responses (e.g., assessed as a frequency ofantigen specific IFN gamma secreting cells and as a frequency ofTNF-α/IFN-γ secreting polyfunctional T cells, which are the desiredimmunological subtype for fighting cancer). Most notably, targeting CEAto the endosomal system using the modifications presented herein alsostimulated both the numbers of IFN-γ secreting CD8 T cells as well asthe polyfunctional variety of these cells. As will be readilyappreciated, such findings are relevant to other TAA such as mesothelin,PSCA, and urokinase plasminogen activator receptor, GPI-anchoredproteins encoded by pathogens, or even GPI-anchored proteins againstwhich an immunosuppressive response is desired like in autoimmunitythrough the inclusion of immune inhibitory factors (like IL-10, TGF-β,etc.). Further suitable GPI-anchored proteins and attachment signals canbe readily identified using bioinformatics analysis (see e.g.,Bioinformatics 2005, Vol. 21 no. 9 2005, pages 1846-1852; BMCBioinformatics 2008, 9:392), while known GPI-anchored proteins can beretrieved from various publically accessible databases (e.g., URL:uniprot.org).

Therefore, the inventors generally contemplate genetic modifications ofGPI-anchored proteins, and especially disease associated GPI-anchoredproteins in which the GPI anchor is replaced by one or moretransmembrane domains, and in which the modified protein furtherincludes a trafficking element that directs the so generated recombinanthybrid protein to a sub-cellular location favoring MHC-II presentation,and especially the recycling endosome, sorting endosome, or lysosome.Most typically, but not necessarily, the replacement of the GPI anchorcan be effected by either removal of the GPI anchor signal sequence orby modification of the GPI anchor signal sequence (e.g., replacement ofone or more amino acids) to abolish or render GPI modification lessefficient. In less preferred aspects, however, GPI modification may alsobe done by adding a peptide linker to the C-terminus of the protein,followed by a transmembrane domain, or by adding a transmembrane domainto the C-terminus of the protein (here: without intervening linker).

With respect to contemplated transmembrane proteins, it should berecognized that many domains are deemed suitable herein, and that themodified protein may contain from one or more (e.g., two, three, four,six, etc.) transmembrane domains. For example, various receptor tyrosinekinases, cytokine receptors, receptor guanylyl cyclases, and receptorserine/threonine protein kinases contain a single transmembrane domain.In other examples, certain ion channels and adenylcyclases have sixtransmembrane domains, and selected cell surface receptors compriseseven transmembrane domains. Exemplary transmembrane proteins includethe insulin receptor, the insulin-like growth factor receptor, the humangrowth hormone receptor, various glucose transporters, the transferrinreceptor, the epidermal growth factor receptor, the LDL receptor, theleptin receptor, various interleukin receptors (e.g., IL-1 receptor,IL-2 receptor, etc.).

Most typically, contemplated transmembrane domains will include about 20consecutive hydrophobic amino acids that may be followed by chargedamino acids. There are numerous transmembrane domains known in the art,and all of them are deemed suitable for use herein. For example,contemplated transmembrane domains can comprise comprises thetransmembrane region(s) of the alpha, beta, or zeta chain of the T-cellreceptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137,CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB(CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160,CD19, IL2Ry, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6,VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM,CD11b, ITGAX, CD11 c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2,DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CEACAM1, CRTAM, Ly9 (CD229),CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, orPAG/Cbp.

Where multiple transmembrane domains are desired, it should be notedthat the hybrid protein may have at least two, three, four, or five, orsix transmembrane domains (or regions that include at least about 10 to35 more preferably about 15 to 30 or 20 to 25 amino acid residues andhave at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100% homology witha known transmembrane domain. As will be readily appreciated,transmembrane domains can be identified by computational analysis ofknown amino acid sequences using prediction methods analyzing secondarystructure, hydrophobicity, and/or topology of a protein in question (seee.g., Biochemistry (1994)33: 3038-3049). Moreover, where contemplatedhybrid proteins have multiple transmembrane domains, individualtransmembrane domains will typically be coupled to each other viaintra-/extracellular loops, which may be formed from about 5-100 aminoacids. Synthetic transmembrane domains may comprise repeat units ofhydrophobic amino acids (e.g., F, W, V). Lastly, it should beappreciated that while use of a transmembrane domain is generallypreferred, the transmembrane domain may also be omitted in at least someembodiments.

With respect to suitable trafficking elements, it is contemplated thatpreferred elements include a CD1b leader sequence, a CD1a tail, a CD1ctail, and a LAMP1-transmembrane sequence. For example, lysosomaltargeting can be achieved using a LAMP1-TM (transmembrane) sequence,while recycling endosomes can be targeted via the CD1a tail targetingsequence, and sorting endosomes can be targeted via the CD1c tailtargeting sequence as is shown in more detail further below.

In particular embodiments, the immunogenic peptides of the inventionfurther comprise an amino acid sequence (or another organic compound)facilitating uptake of the peptide into (late) endosomes for processingand presentation within CD1d determinants. The late endosome targetingis mediated by signals present in the cytoplasmic tail of proteins andcorrespond to well-identified peptide motifs such as the dileucine-basedmotifs a tyrosine-based motif or the so-called acidic cluster motif. Thesymbol o represents amino acid residues with a bulky hydrophobic sidechains such as Phe, Tyr and Trp. The late endosome targeting sequencesallow for processing and efficient presentation of the antigen-derived Tcell epitope by CD1d molecules. Such endosomal targeting sequences arecontained, for example, within the gp75 protein (J Cell Biol 130,807-820), the human CD3 gamma protein, the HLA-BMβ. (J. Immunol.(1996)157, 1017-1027), the cytoplasmic tail of the DEC205 receptor (JCell Biol (2000)151, 673-683). Other examples of peptides which functionas sorting signals to the endosome are disclosed in the review ofBonifacio and Traub (Annu. Rev. Biochem. (2003)72, 395-447. See alsoFront Biosci 2009.

Viewed from a different perspective, the hybrid proteins contemplatedherein will include a trafficking signal that will result inpreferential trafficking (e.g., at least 70%, more typically at least80%, and most typically at least 90% of all expressed hybrid proteinsare found in the targeted sub-cellular compartment) of the hybridprotein to the desired sub-cellular location. Therefore, in contemplatedaspects of the inventive subject matter, signal and/or leader peptidesmay be used for trafficking neoepitopes and/or polytopes to theendosomal or lysosomal compartment. Thus, it should also be recognizedthat targeting presequences and/or targeting peptides can be employed.The presequences of the targeting peptide may be added to the N-terminusand/or C-terminus and typically comprise between 6-136 basic andhydrophobic amino acids. In case of peroxisomal targeting, the targetingsequence may be at the C-terminus. Other signals (e.g., signal patches)may be used and include sequence elements that are separate in thepeptide sequence and become functional upon proper peptide folding.

In addition, protein modifications like glycosylations can inducetargeting. Among other suitable targeting signals, the inventorscontemplate peroxisome targeting signal 1 (PTS1), a C-terminaltripeptide, and peroxisome targeting signal 2 (PTS2), which is anonapeptide located near the N-terminus. In addition, sorting ofproteins to endosomes and lysosomes may also be mediated by signalswithin the cytosolic domains of the proteins, typically comprisingshort, linear sequences. Some signals are referred to as tyrosine-basedsorting signals or as dileucine-based signals. All of these signals arerecognized by components of protein coats peripherally associated withthe cytosolic face of membranes. Other known signals are recognized withcharacteristic specificity by the adaptor protein (AP) complexes AP-1,AP-2, AP-3, and AP-4, whereas still other signals are recognized byanother family of adaptors known as GGAs.

It should further be appreciated that the transmembrane domain(s) andthe trafficking sequences may in some embodiments be coupled to theantigen via a linker, which is preferably a flexible linker comprisesbetween 5 and 50 amino acids. For example, contemplated linkers includeflexible glycine/serine linkers, and rigid linkers. There are variouslinker sequences known on the art (see e.g., Adv Drug Deliv Rev. 2013Oct. 15; 65(10): 1357-1369), and all of these linkers are deemedsuitable for use herein.

In still further contemplated embodiments, the recombinant hybridprotein may also be modified to facilitate trafficking to or retentionin the cytosolic compartment (which may not necessarily require one ormore specific sequence elements). For example, in at least some aspects,N- or C-terminal cytoplasmic retention signals may be added, includingcytoplasmic retention signals of SNAP-25, syntaxin, synaptoprevin,synaptotagmin, vesicle associated membrane proteins (VAMPs), synapticvesicle glycoproteins (SV2), high affinity choline transporters,neurexins, voltage-gated calcium channels, acetylcholinesterase, andNOTCH. Thus, it should be appreciated that peptides can be routed tospecific cellular compartments to so achieve preferential or evenspecific presentation via MHC-I or MHC-II.

Still further contemplated increases in antigenicity of the recombinanthybrid proteins presented herein can be achieved by(mono/poly)ubiquitinylation via addition of one or more recombinantubiquitination motifs. There are numerous motifs known in the art, andall of these are deemed suitable for use herein (see e.g., Proteins 2010Feb. 1; 78(2): 365-380).

While not limiting to the inventive subject matter, the modifiedproteins will generally be expressed in vitro or in vivo from anappropriately constructed recombinant nucleic acid, and especiallysuitable recombinant nucleic acid include plasmids and viral nucleicacids. Where a viral nucleic acid is employed, it is particularlypreferred that the nucleic acid is delivered via infection of thepatient or patient cells by the virus. Therefore, contemplatedcompositions may be administered as a recombinant viral, yeast, orbacterial vaccine, or as a gemisch of multiple (typically distinct)proteins or hybrid polypeptides. Among other contemplated viralexpression vectors and viruses, adenoviral vectors and viruses (e.g.,E2b deleted AdV) are especially contemplated.

Viewed from a different perspective, it should be appreciated that thecompositions and methods presented herein will deliver an otherwisepoorly immunogenic antigen in a manner that facilitates MHC-IIpresentation. Indeed, such modified proteins can advantageously betailored to achieve various specific immune reactions, including anenhanced CD4⁺ immune response and surprisingly an enhanced CD8⁺ immuneresponse. In addition, contemplated hybrid proteins may be co-expressedor co-administered with other immune stimulatory compositions (that maypreferably be encoded on the same recombinant nucleic acid). Forexample, a recombinant nucleic acid may be constructed that includes anexpression cassette that encodes one or more of a co-stimulatorymolecule, an immune stimulatory cytokine, and a protein that interfereswith or down-regulates checkpoint inhibition. Suitable co-stimulatorymolecules include OX40L, 4-1BBL, CD80, CD86, CD30, CD40, CD3OL, CD4OL,ICOS-L, B7-H3, B7-H4, CD70, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A,ICAM-1, and LFA3, and suitable immune stimulatory cytokines includeIL-2, IL-12, IL-15, IL-15 super agonist (ALT803), IL-21, IPS1, and LMP1.In further contemplated aspects, preferred proteins that interfere withcheckpoint inhibition include antibodies or antagonists of CTLA-4, PD-1,TIM1 receptor, 2B4, or CD160. Likewise, additionally encoded signalsinclude protein adjuvants like calreticulin or HMBG proteins (orfragments thereof).

Therefore, in exemplary preferred aspects of the inventive subjectmatter, cancer immune therapy may uses a recombinant adenovirus that hasas payload in which a modified TAA has a deleted or otherwisenon-functional GPI anchor sequence and that also includes atransmembrane domain and a trafficking signal as described above.Regardless of the type of recombinant virus it is contemplated that thevirus may be used to infect patient (or non-patient) cells ex vivo or invivo. For example, the virus may be injected subcutaneously orintravenously, or may be administered intranasaly or via inhalation toso infect the patients cells, and especially antigen presenting cells.Alternatively, immune competent cells (e.g., NK cells, T cells,macrophages, dendritic cells, etc.) of the patient (or from anallogeneic source) may be infected in vitro and then transfused to thepatient. Alternatively, immune therapy need not rely on a virus but maybe effected with nucleic acid transfection or vaccination using RNA orDNA, or other recombinant vector that leads to the expression of theneoepitopes (e.g., as single peptides, tandem mini-gene, etc.) indesired cells, and especially immune competent cells.

Most typically, the desired nucleic acid sequences (for expression fromvirus infected cells) are under the control of appropriate regulatoryelements well known in the art. For example, suitable promoter elementsinclude constitutive strong promoters (e.g., SV40, CMV, UBC, EF1A, PGK,CAGG promoter), but inducible promoters are also deemed suitable for useherein, particularly where induction conditions are typical for a tumormicroenvironment. For example, inducible promoters include thosesensitive to hypoxia and promoters that are sensitive to TGF-β or IL-8(e.g., via TRAF, JNK, Erk, or other responsive elements promoter). Inother examples, suitable inducible promoters include thetetracycline-inducible promoter, the myxovirus resistance 1 (Mx1)promoter, etc. Alternatively, it should be appreciated that cancervaccine compositions need not be limited to adenovirus constructs asdescribed above, but may include recombinant yeast and bacteria as wellas recombinant protein coupled to a carrier.

Where the expression construct is a viral expression construct (e.g., anadenovirus, and especially AdV with E1 and E2b deleted), it iscontemplated that the recombinant viruses may then be individually or incombination used as a therapeutic vaccine in a pharmaceuticalcomposition, typically formulated as a sterile injectable compositionwith a virus titer of between 10⁶-10¹³ virus particles, and moretypically between 10⁹-10¹² virus particles per dosage unit.Alternatively, virus may be employed to infect patient (or other HLAmatched) cells ex vivo and the so infected cells are then transfused tothe patient. In further examples, treatment of patients with the virusmay be accompanied by allografted or autologous natural killer cells orT cells in a bare form or bearing chimeric antigen receptors expressingantibodies targeting neoepitope, neoepitopes, tumor associated antigensor the same payload as the virus. The natural killer cells, whichinclude the patient-derived NK-92 cell line, may also express CD16 andcan be coupled with an antibody.

Where desired, additional therapeutic modalities may be employed whichmay be neoepitope based (e.g., synthetic antibodies against neoepitopesas described in WO 2016/172722), alone or in combination with autologousor allogenic NK cells, and especially haNK cells or taNK cells (e.g.,both commercially available from NantKwest, 9920 Jefferson Blvd. CulverCity, Calif. 90232). Where haNK or taNK cells are employed, it isparticularly preferred that the haNK cell carries a recombinant antibodyon the CD16 variant that binds to a neoepitope of the treated patient,and where taNK cells are employed it is preferred that the chimericantigen receptor of the taNK cell binds to a neoepitope of the treatedpatient. The additional treatment modality may also be independent ofneoepitopes, and especially preferred modalities include cell-basedtherapeutics such as activated NK cells (e.g., aNK cells, commerciallyavailable from NantKwest, 9920 Jefferson Blvd. Culver City, Calif.90232), and non cell-based therapeutics such as chemotherapy and/orradiation. In still further contemplated aspects, immune stimulatorycytokines, and especially IL-2, IL15, and IL-21 may be administered,alone or in combination with one or more checkpoint inhibitors (e.g.,ipilimumab, nivolumab, etc.).

Similarly, it is still further contemplated that additionalpharmaceutical intervention may include administration of one or moredrugs that inhibit immune suppressive cells, and especially MDSCs Tregs,and M2 macrophages. Thus, suitable drugs include IL-8 or interferon-γinhibitors or antibodies binding IL-8 or interferon-γ, as well as drugsthat deactivate MDSCs (e.g., NO inhibitors, arginase inhibitors, ROSinhibitors), that block development of or differentiation of cells toMDSCs (e.g., IL-12, VEGF-inhibitors, bisphosphonates), or agents thatare toxic to MDSCs (e.g., gemcitabine, cisplatin, 5-FU). Likewise, drugslike cyclophosphamide, daclizumab, and anti-GITR or anti-OX40 antibodiesmay be used to inhibit Tregs.

To trigger overexpression or transcription of stress signals, it is alsocontemplated that the chemotherapy and/or radiation for the patient maybe done using a low-dose regimen, preferably in a metronomic fashion.For example, it is generally preferred that such treatment will usedoses effective to affect at least one of protein expression, celldivision, and cell cycle, preferably to induce apoptosis or at least toinduce or increase the expression of stress-related genes (andparticularly NKG2D ligands). Thus, in further contemplated aspects, suchtreatment will include low dose treatment using one or morechemotherapeutic agents. Most typically, low dose treatments will be atexposures that are equal or less than 70%, equal or less than 50%, equalor less than 40%, equal or less than 30%, equal or less than 20% , equalor less than 10%, or equal or less than 5% of the LD₅₀ or IC₅₀ for thechemotherapeutic agent. Additionally, where advantageous, such low-doseregimen may be performed in a metronomic manner as described, forexample, in U.S. Pat. Nos. 7,758,891, 7,771,751, 7,780,984, 7,981,445,and 8,034,375.

With respect to the particular drug used in such low-dose regimen, it iscontemplated that all chemotherapeutic agents are deemed suitable. Amongother suitable drugs, kinase inhibitors, receptor agonists andantagonists, anti-metabolic, cytostatic and cytotoxic drugs are allcontemplated herein. However, particularly preferred agents includethose identified to interfere or inhibit a component of a pathway thatdrives growth or development of the tumor. Suitable drugs can beidentified using pathway analysis on omics data as described in, forexample, WO 2011/139345 and WO 2013/062505. Most notably, so achievedexpression of stress-related genes in the tumor cells will result insurface presentation of NKG2D, NKP30, NKP44, and/or NKP46 ligands, whichin turn activate NK cells to specifically destroy the tumor cells. Thus,it should be appreciated that low-dose chemotherapy may be employed as atrigger in tumor cells to express and display stress related proteins,which in turn will trigger NK-cell activation and/or NK-cell mediatedtumor cell killing. Additionally, NK-cell mediated killing will beassociated with release of intracellular tumor specific antigens, whichis thought to further enhance the immune response.

EXAMPLES

The inventor prepared various adenoviral expression constructscontaining null payload (Group1), CEA payload (Group 2), CEA-CD1cpayload (Group 3), CEA-LAMP1 payload (Group 4), CEA-CD1a payload (Group5), and mice were immunized following a biweekly prime/boost regimenusing 10¹⁰ viral particles per injection as shown FIG. 2A. All mice wereeuthanized at day 35 and splenocytes and peripheral blood werecollected. FIG. 2B depicts schematically recombinant constructs used inthe scheme of FIG. 2A.

FIG. 3 provides exemplary results with respect to CD4+ splenocytes. Theleft panel illustrates the fraction of ICS stim IFNγ⁺ CD4⁺ cells thatwere observed in response to media (left), an unrelated peptide (SIV nefpeptide, middle), and CEA peptide (right). As expected, no significantstimulation of CD4⁺ cells was found with media and unrelated protein.However, when exposed to CEA peptide, all CEA bearing adenoviralconstructs resulted in a significant response, with a substantiallyenhanced response in those where trafficking was forced to theendo-/lysosomal compartments. Similarly, the fraction of IFNγ⁺TNFα⁺CD4⁺cells was significantly increased with where trafficking was forced tothe endo-/lysosomal compartments (right panel).

Even more notably, when the same experiments were used to observe CD8⁺cells, the inventor discovered that IFNγ⁺ CD8⁺ as well as IFNγ⁺ TNFα⁺CD8⁺ cells were significantly increased where trafficking was forced tothe endo-/lysosomal compartments as can be taken from FIG. 4. Suchenhancement was particularly pronounced vis-á-vis adenoviral delivery ofCEA peptide alone. Indeed, the significant increase in reactive CD8⁺cells is particularly unexpected in cases where the recombinant hybridprotein was targeted to the endosomal and/or lysosomal pathway (which istypically the route for MHC-I presentation). While not limiting to theinventive subject matter, it is contemplated that the recombinant hybridproteins presented herein will advantageously be subject to across-presentation type antigen processing. Thus, it should beappreciated that contemplated systems and methods not only substantiallyenhance an immune response against an otherwise difficult to targetantigen (GPI-anchored antigen), but also increase the fraction ofpolyfunctional CD4+ and CD8+ cells. Advantageously, all immunizedanimals were also able to produce significant quantities of antibodiesas is shown in the graph depicting anti-CEA ELISA in FIG. 5.

The following sequences were used in the above experiments, with theleader peptide shown underlined, transmembrane domain shown in boldtypeface, and endosomal targeting sequences shown in italics. Allsequences were subcloned into and expressed from an E2b deletedadenovirus AdV that was injected subcutaneously into mice following theimmunization schedule of FIG. 2A.

>CEA1 (SEQ ID NO: 1): MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREITYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNELSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGADLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVALI >CEA1-CD1a (SEQ ID NO: 2):MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREITYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNELSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGADLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVMLIPIAVGGALAGLVLIVLIAYLIG RKRCFC >CEA1-CD1c (SEQ ID NO: 3):MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREITYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNELSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGADLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVMLIPIAVGGALAGLVLIVLIAYLIG KKHCSYQDIL >CEA1-LAMP1 (SEQ ID NO: 4):MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREITYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNELSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGADLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVMLIPIAVGGALAGLVLIVLIAYLIG R KRSHAGYQTI

As used herein, the term “administering” a pharmaceutical composition ordrug refers to both direct and indirect administration of thepharmaceutical composition or drug, wherein direct administration of thepharmaceutical composition or drug is typically performed by a healthcare professional (e.g., physician, nurse, etc.), and wherein indirectadministration includes a step of providing or making available thepharmaceutical composition or drug to the health care professional fordirect administration (e.g., via injection, infusion, oral delivery,topical delivery, etc.). Most preferably, the recombinant virus isadministered via subcutaneous or subdermal injection. However, in othercontemplated aspects, administration may also be intravenous injection.Alternatively, or additionally, antigen presenting cells may be isolatedor grown from cells of the patient, infected in vitro, and thentransfused to the patient. Therefore, it should be appreciated thatcontemplated systems and methods can be considered a complete drugdiscovery system (e.g., drug discovery, treatment protocol, validation,etc.) for highly personalized cancer treatment.

The recitation of ranges of values herein is merely intended to serve asa shorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . .and N, the text should be interpreted as requiring only one element fromthe group, not A plus N, or B plus N, etc.

1. A recombinant hybrid protein, comprising: an antigen portion, coupledto at least one transmembrane domain and a trafficking element; whereinthe antigen portion is at least a portion of a GPI-anchored protein; andwherein the trafficking element directs the recombinant hybrid proteinto a sub-cellular location selected from the group consisting of arecycling endosome, a sorting endosome, and a lysosome.
 2. The hybridprotein of claim 1 wherein the GPI-anchored protein is CEA, PSCA,mesothelin, or urokinase plasminogen activator receptor.
 3. The hybridprotein of claim 1 wherein the GPI-anchored protein is a non-cancerdisease associated protein, optionally a variant surface protein ofTrypanosoma brucei or a prion protein.
 4. The hybrid protein of claim 1wherein the antigen portion lacks a functional GPI anchoring signal. 5.The hybrid protein of claim 1 wherein the transmembrane domain comprisesat least a portion of a transmembrane domain of an alpha, beta, or zetachain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CDS, CD8(e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80,CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18),ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7,NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1,CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE,CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11 c, ITGB1, CD29,ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4),CD84, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D),SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8),SELPLG (CD162), LTBR, or PAG/Cbp, and/or wherein the transmembranedomain is bound to the C-terminus of the antigen portion, and/or whereinthe transmembrane domain is coupled to the C-terminus of the antigenportion via a peptide linker. 6-7. (canceled)
 8. The hybrid protein ofclaim 1 wherein the trafficking element comprises an endosomaltrafficking element of CD1a, CD1c, or Lampl and/or wherein thetrafficking element is bound in-frame to the C-terminus of thetransmembrane domain.
 9. (canceled)
 10. A recombinant nucleic acid,comprising: a sequence segment encoding the hybrid protein of claim 1,the sequence segment operably linked to a promoter to drive expressionof the hybrid protein.
 11. The recombinant nucleic acid of claim 10wherein the nucleic acid is a viral expression vector or wherein theviral expression vector is an adenoviral expression vector, optionallyhaving a deleted E1 and E2b gene.
 12. (canceled)
 13. The recombinantnucleic acid of claim 10 wherein the promoter is a constitutive promoteror an inducible promoter, optionally inducible by hypoxia, IFN-gamma, orIL-8.
 14. (canceled)
 15. The recombinant nucleic acid of claim 10wherein the recombinant nucleic acid further comprises a sequence thatencodes at least one of a co-stimulatory molecule, an immune stimulatorycytokine, a protein that interferes with or down-regulates checkpointinhibition, and an adjuvant polypeptide, wherein the co-stimulatorymolecule is selected from the group consisting of OX4OL, 4-1BBL, CD80,CD86, CD30, CD40, CD3OL, CD40L, ICOS-L, B7-H3, B7-H4, CD70, GITR-L,TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, and LFA3, wherein the immunestimulatory cytokine is selected from the group consisting of IL-2,IL-12, IL-15, IL-15 super agonist (ALT803), IL-21, IPS1, and LMP1,and/or wherein the protein that interferes is an antibody or anantagonist of CTLA-4, PD-1, TIM1 receptor, 2B4, or CD160. 16-18.(canceled)
 19. The recombinant nucleic acid of claim 15 wherein theadjuvant polypeptide is calreticulin or a portion with adjuvant activitythereof, or wherein the adjuvant polypeptide is HMGB1 or a portion withadjuvant activity thereof
 20. A recombinant virus comprising therecombinant nucleic acid of claim
 10. 21. The recombinant virus of claim20 wherein the recombinant virus is a replication deficient virus, andoptionally wherein the recombinant virus is an adenovirus having adeleted E1 and E2b gene.
 22. A recombinant antigen presenting cellcomprising the recombinant nucleic acid of claims 10 or expressing thehybrid protein of claim
 1. 23. A method of increasing antigenicity of aGPI-anchored protein, comprising: modifying the protein portion of theGPI-anchored protein to include at least one transmembrane domain and atrafficking element; and wherein the trafficking element directs themodified protein to a sub-cellular location selected from the groupconsisting of a recycling endosome, a sorting endosome, and a lysosome.24. The method of claim 23 wherein the GPI-anchored protein is CEA,PSCA, mesothelin, or urokinase plasminogen activator receptor.
 25. Themethod of claim 23 wherein the GPI-anchored protein is a non-cancerdisease associated protein, optionally a variant surface protein ofTrypanosoma brucei or a prion protein.
 26. The method of claim 23wherein the antigen portion lacks a functional GPI anchoring signal. 27.The method of claim 23 wherein the transmembrane domain comprises atleast a portion of a transmembrane domain of an alpha, beta, or zetachain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CDS, CD8(e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80,CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18),ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7,NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1,CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE,CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11 c, ITGB1, CD29,ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4),CD84, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D),SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8),SELPLG (CD162), LTBR, or PAG/Cbp and/or wherein the transmembrane domainis bound to the C-terminus of the antigen portion and/or wherein thetransmembrane domain is coupled to the C-terminus of the antigen portionvia a peptide linker. 28-29. (canceled)
 30. The method of claim 23wherein the trafficking element comprises an endosomal traffickingelement of CD1a, CD1c, or Lamp1, or wherein the trafficking element isbound in-frame to the C-terminus of the transmembrane domain. 31-40.(canceled)